There are two long-term objectives in this proposal. One is to better comprehend how certain bacterial and plant protein toxins exploit pathways of membrane traffic to enter the cytosol of eukaryotic cells. Information about the entry process is important to understand how pathogenic toxins work and also may provide insight into basic cellular processes. The second objective is to capitalize on the properties of protein toxins to develop applications of therapeutic value. There are four specific aims: AIM 1, analysis of toxin entry into CHO cell mutants of the ldlF group. ldlF mutants express a temperature-sensitive defect in epsilon-COP, a subunit of non-clathrin coatomers. These cells serve as a model system to investigate whether toxin access to the cytosol requires epsilon-COP. AIM 2, the interaction of precleaved exotoxin A (ETA) with cells. ETA must be proteolytically cleaved to enter the cytosol. We are studying here the consequences of precleaving the toxin before adding it to cells. AIM 3, the effect of covalently attaching apyrase to ETA. The objective of this aim is to determine whether ETA enters the endoplasmic reticulum before entering the cytosol. If it does, then ETA should carry attached apyrase to the endoplasmic reticulum, which should have detectable consequences. AIM 4, studies of a variant of ETA that has a free sulfhydryl near the carboxyl terminus. ETA with a cysteine residue inserted near the carboxyl terminus has much-reduced cytotoxicity. The objective of this aim is to understand why.